The long-term goal is to understand how synapse structure in the adult central nervous system supports learning and memory. We study hippocampal long-term potentiation (LTP), a form of synaptic plasticity that is viewed as a cellular substrate for learning and memory. The focus is on dendritic spines, the tiny protrusions that host more than 90% of excitatory synapses in the brain, are modified during LTP, learning, and memory, and are severely distorted in a variety of neurological disorders. Recently, we have shown that initially saturated LTP can be subsequently augmented if more than 1.5 hours elapse between episodes of LTP induction, with 100% success achieved in adult mouse hippocampus after a 4 hour interval. These findings support the hypothesis that spacing episodes of LTP engages mechanisms that might underlie the advantage of spaced over massed learning. Using 3D reconstruction from serial section electron microscopy (3DEM), we have discovered several changes in synapse structure that manifest over time after the initial saturation of LTP. Both in vivo and in vitro, many synapses had nascent zones, dynamic edge regions that have a postsynaptic density but lack the presynaptic vesicles normally found at active zones. Nascent zones rapidly acquired presynaptic vesicles, thereby converting to active zones however by 30 min. By 2 hr., both nascent and active zones were enlarged, and the greatest synapse enlargement occurred on spines that contained smooth endoplasmic reticulum (SER) and polyribosomes. We will test the hypothesis that the capacity to modify synaptic structure is initially saturated by LTP, and time is required for synapses to recover or grow in preparation for later augmentation. We will verify successful LTP induction with physiology following various pharmacological and genetic manipulations in mature mouse hippocampus prior to performing the more time consuming 3DEM and immunolabeling. We aim to test the following hypotheses regarding mechanisms of augmentation: 1) That receptors and presynaptic docking sites must accumulate at synapses enlarged after the initial saturation of LTP. 2) That SER-dependent synapse growth and spine clustering serve the augmentation of LTP. 3) That protein synthesis-dependent growth of synapses is required for augmentation of LTP. 4) That absence of candidate molecules involved in building or stabilizing synapses disrupts saturation or augmentation of LTP. Upon completion of these aims we will know which elements of structural synaptic plasticity are integral to the augmentation of LTP. Outcomes will provide new understanding of the mechanisms of nascent zone conversion, synapse growth, and spine clustering, and whether these processes are coupled to SER expansion, local protein synthesis, and synapse adhesion in preparation for the subsequent augmentation of initially saturated LTP in the mature mouse brain. The results should ultimately inform the development of new strategies to repair dysfunctional synaptic circuits.